In article <C8vqIC.KF8 at mentor.cc.purdue.edu>, pmiguel at bilbo.bio.purdue.edu writes...
>In article <19JUN199308260889 at aardvark.ucs.uoknor.edu>, broe at aardvark.ucs.uoknor.edu (Bruce Roe) writes:
>>>>I have a tendency to think about bands in all 4 lanes as being due
>>to "template compression" i.e. secondary structure, since "product
>>compression" also is due to secondary structure ( fold back )
>>in the synthesized strand.
>>> Yeah, but calling template secondary structure "compression" is
>confusing. "Product compression" is seen as bands compressed together on
>the sequencing gel, thus the name. Extremely bad compression could look
>like or be accompanied by what you call "template compression" -- bands
>across all four lanes. I call this "fall-off" because it is so refered to in
>the Sequenase manual, because it makes sense to me and, probably more
>important isn't easy to confuse with "compression."
> I'm not just quibbling over nomenclature here. If you have a compression
>you need to run a gel with formamide in it or do rxns with dITP or dazaG
>replacing dG. If you have fall-off you need to increase the temperature of
>your extensions or use SSB to eliminate template secondary structure.
>>>Phillip
Phillip,
Whatever works and makes sense to you is fine by me. But it
is curious that one takes for "fact" what they read in the product literature:
"I call this "fall-off" because it is so refered to in the Sequenase manual"
although I believe Tabor and Richardson might have implied "fall off" in
their PNAS paper.
I'd also really like to know what's on the 3' end of those "fall off"
(as you put it) or "template associated compression" (as I put it) bands.
If these bands truly are due to "fall off" (which is caused by the enzyme
pausing at regions of template secondary structure), then the vast majority
of the products would have a 3' deoxynucleotide. If however, these "false
terminations" are due to "mis-incorporation" as the enzyme trys to continue
through regions of strong secondary structure, then it might be that
the observed "false terminations" are due to mis-incorporation of a
dideoxynucleotide and the subsequent dissociation of the enzyme from
the template.
Anyone got any clues from experiments they've done or from the
primary literature?
Cheers......bruce
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\ Bruce A. Roe Professor of Chemistry and Biochemistry /
/ University of Oklahoma INTERNET: BROE at aardvark.ucs.uoknor.edu \
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